EP2336342A1 - Détoxication avec des agents réducteurs - Google Patents

Détoxication avec des agents réducteurs Download PDF

Info

Publication number
EP2336342A1
EP2336342A1 EP09180193A EP09180193A EP2336342A1 EP 2336342 A1 EP2336342 A1 EP 2336342A1 EP 09180193 A EP09180193 A EP 09180193A EP 09180193 A EP09180193 A EP 09180193A EP 2336342 A1 EP2336342 A1 EP 2336342A1
Authority
EP
European Patent Office
Prior art keywords
fermentation
reducing agent
dithionite
added
sulphite
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09180193A
Other languages
German (de)
English (en)
Inventor
Björn Alriksson
Adnan Cavka
Leif JÖNSSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sekab E Technology AB
Original Assignee
Sekab E Technology AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sekab E Technology AB filed Critical Sekab E Technology AB
Priority to EP09180193A priority Critical patent/EP2336342A1/fr
Priority to PL10795363T priority patent/PL2516659T3/pl
Priority to CA2783200A priority patent/CA2783200C/fr
Priority to AP2012006373A priority patent/AP3315A/xx
Priority to CA3035054A priority patent/CA3035054C/fr
Priority to US13/517,284 priority patent/US8795997B2/en
Priority to BR112012015508-3A priority patent/BR112012015508B1/pt
Priority to PCT/EP2010/070127 priority patent/WO2011080129A2/fr
Priority to EP10795363.0A priority patent/EP2516659B1/fr
Priority to CN201080064339.8A priority patent/CN102770547B/zh
Priority to EP13194379.7A priority patent/EP2703493B1/fr
Publication of EP2336342A1 publication Critical patent/EP2336342A1/fr
Priority to ZA2012/04178A priority patent/ZA201204178B/en
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a method for decreasing fermentation inhibitory effects of a slurry or hydrolysate during fermentation.
  • Lignocellulosic residues from forestry and agriculture are attractive as feedstocks, since they are abundant, relatively inexpensive, and are not used for food.
  • Lignocellulose consists mainly of lignin and two classes of polysaccharides, cellulose and hemicellulose. The polysaccharides can be hydrolyzed to sugars and converted to various fermentation products, such as bioalcohols, in processes based on biocatalysts, such as the industrially important baker's yeast (Saccharomyces cerevisiae).
  • the hydrolysis of cellulose is typically preceded by a pretreatment, in which the hemicellulose is degraded and the cellulose is made increasingly accessible to cellulolytic enzymes.
  • the pretreatment process typically generates fermentation inhibitors, such as phenolic compounds, aliphatic acids, and furan aldehydes, which have a negative effect on the efficiency of the fermentation process.
  • fermentation inhibitors such as phenolic compounds, aliphatic acids, and furan aldehydes
  • the inventors have realized that there is a need in the art for improved methods to overcome problems with fermentation inhibition in the manufacture of fermentation products from cellulosic material.
  • a method for decreasing the fermentation inhibition in a process for producing a target chemical from a pretreated cellulosic material comprising enzymatic hydrolysis of the pretreated cellulosic material and fermentation of hydrolysed material, wherein the fermentation inhibitory properties of the material subjected to fermentation is decreased by an addition of at least one reducing agent to the pretreated material or hydrolysed material.
  • dithionite for decreasing the fermentation inhibitory properties of a material being subjected to simultaneous enzymatic hydrolysis and fermentation or a hydrolysate derived from enzymatic hydrolysis being subjected to fermentation.
  • a method for decreasing the fermentation inhibition in a process for producing a target chemical from a pretreated cellulosic material comprising enzymatic hydrolysis of the pretreated cellulosic material and fermentation of hydrolysed material, wherein the fermentation inhibitory properties of the material subjected to fermentation is decreased by an addition of at least one reducing agent to the pretreated material or hydrolysed material.
  • Frermentation is a process known to the skilled person, and is usually performed by microorganisms.
  • “Fermentation inhibition” refers to a negative effect on a fermentation reaction, e.g. a decreasing of the rate of the fermentation reaction or the total amount of target product produced in the fermentation reaction. "Decreasing the fermentation inhibition” thus refers to decreasing such negative effect. Consequently, decreasing the fermentation inhibition may be detoxification or conditioning of a material subjected to fermentation, i.e. decreasing the effect of one or more properties of the material subjected to fermentation, which properties are inhibiting the fermenting organism's conversion of a substrate to the target chemical. For example, “decreasing the fermentation inhibition” may be increasing the saccharide consumption rate, such as the glucose consumption rate, increasing the total amount of target chemical produced during fermentation, increasing the target chemical yield on consumed saccharide during fermentation, i.e. increasing the number of target chemical molecules produced by each consumed saccharide molecule, or increasing the volumetric target chemical productivity, e.g. measured as (g target chemical ⁇ L -1 ⁇ h -1 )
  • a "cellulosic material” refers to any material comprising cellulose and/or hemicellulose.
  • the cellulosic material may be lignocellulosic material, i.e. material comprising cellulose, lignin and possibly hemicellulose.
  • the lignocellulosic material may for example be wood residues or forestry residues, such as wood chips, sawmill or paper mill discards, or agricultural residues, such as sugarcane bagass.
  • Pretreated cellulosic material refers to cellulosic material that has been pretreated in order to modify its properties such that the cellulose becomes more accessible during subsequent hydrolysis.
  • the pretreatment may involve one or several pretreatment methods known to the skilled man.
  • the pretreatment may be acid pretreatment or alkali pretreatment.
  • the pretreatment may be impregnation, which refers to impregnating of the cellulosic material with an impregnation fluid, followed by heating.
  • the fluid may be an acid solution, such as a mineral acid solution.
  • the impregnation may also be performed with a gas, such as a SO 2 -gas or CO 2 -gas, or with the combination of a gas with a liquid.
  • the pretreatment may also comprise steaming.
  • Steaming refers to a process used to drive air out from the cellulosic biomass to facilitate further hydrolysis of the cellulose. Steaming is a well-known method for pretreating e.g. lignocellulosic biomass. As another example, the pretreatment may involve steam explosion. Steam explosion is a process that combines steam, rapid pressure release and hydrolysis for rupturing cellulosic fibers.
  • a "target chemical from a pretreated cellulosic material” refers to any chemical that can be prepared from pretreated cellulosic material in a process comprising fermentation.
  • Enzymatic hydrolysis refers to a hydrolysis reaction catalysed by at least one enzyme.
  • the at least one enzyme may be at least one saccharification enzyme, which refers to at least one enzyme that can convert or hydrolyse cellulosic material into fermentable saccharides, such as monosaccharides and/or disaccharides.
  • saccharification enzymes may be glycosidases, which hydrolyse polysaccharides.
  • glycosidases examples include cellulose-hydrolysing glycosidases, such as cellulases, endoglucanases, exoglucanases, cellobiohydrolases and ⁇ -glucosidases, hemicellulose hydrolysing glycosidases, such as xylanases, endoxylanases, exoxylanases, ⁇ -xylosidases, arabinoxylanases, mannanases, galactanases, pectinases and glucuronases, and starch hydrolysing glycosidases, such as amylases, ⁇ -amylases, ⁇ -amylases, glucoamylases, ⁇ -glucosidases and isoamylases, or any enzymes in the group of enzymes found in EC 3.2.1.x, such as EC 3.2.1.4, where EC is the Enzyme Commission number.
  • a "reducing agent” refers to a chemical agent capable of causing the reduction of another substance as it itself is oxidized, i.e. a chemical agent capable of donating an electron in an oxidation-reduction reaction.
  • addition of at least one reducing agent to the pretreated material or hydrolysed material thus refers to addition of at least one reducing agent to a cellulosic material that has already been subjected to pretreatment.
  • the addition of the reducing agent occurs downstream of any pretreatment of cellulosic material in the process for producing a target chemical.
  • the present invention is based on the insight that the addition of a reducing agent to pretreated cellulosic material is an effective approach to overcome obstacles connected with bioconversion of cellulosic material to target chemicals.
  • a dramatic improvement in fermentability can be achieved with a relatively small addition of reducing agent and further, the reducing agent is compatible with enzymes and fermenting organisms such as yeast, thus resulting in little influence on enzyme or yeast performance.
  • detoxification or conditioning of pretreated cellulosic material using a reducing agent according to the first aspect does not require introduction of genetically modified microorganisms in the industrial process.
  • Further benefits of the method according to the first aspect of the invention include that the addition of the reducing agent can be carried out at a pH suitable for fermentation and at room or fermentation temperature, and results in improved fermentability without degradation of fermentable sugars.
  • the enzymatic hydrolysis and fermentation are performed in two separate steps and the fermentation step is performed in a fermentor.
  • a "fermentor" refers to any type of container that may be used for preparing a target chemical by means of fermentation.
  • the cellulosic material may first be subjected to enzymatic hydrolysis to produce free sugars that in a separate process step are fermented into the target chemical.
  • the process for producing a target chemical may be performed as a separate hydrolysis and fermentation (SHF) process.
  • SHF process offers the possibility to perform the enzymatic hydrolysis and fermentation at different process conditions, such as at different pH and temperature.
  • the at least one reducing agent may be added to the fermentor.
  • Addition of the reducing agent to the fermentor is advantageous, since there is no need to perform any additional separate steps for addition of the reducing agent, which could contribute to higher process costs.
  • addition of the reducing agent during fermentation permits full process flexibility, i.e. the general process design does not need to be adapted or amended for decreasing fermentation inhibition since the addition of a reducing agent is performed in the step of fermentation.
  • the reducing agent may be added prior to or after a fermenting organism is added to the fermentor. Further, the reducing agent may be added concurrently with the fermenting organism.
  • the enzymatic hydrolysis and fermentation are performed simultaneously in a fermentor.
  • the process for producing a target chemical may be performed as a simultaneous saccharification and fermentation (SSF) process, in which hydrolysis of cellulosic material is achieved through addition of enzymes, such as cellulase, from external sources, or as a consolidated bioprocess (CBP), in which the biocatalyst that convert the monosaccharides also produces the enzymes that hydrolyze the cellulosic material.
  • SSF simultaneous saccharification and fermentation
  • CBP consolidated bioprocess
  • the at least one reducing agent may be added to the fermentor.
  • the reducing agent may be added at any stage of the fermentation process, e.g. only if and when needed. As described above, the reducing agent may be added prior to or after enzymes and/or a fermenting organism are added to the fermentor. Further, the reducing agent may be added concurrently with the enzymes and/or the fermenting organism.
  • the method according to the first aspect provides for a chemical in situ detoxification of the material subjected to fermentation.
  • the at least one reducing agent is added at a temperature of 20-80 °C, such as 20-75 °C, such as 20-45 °C, such as 28-38 °C.
  • the reducing agent may be added as the material subjected to fermentation has a temperature of 20-45 °C, such as 28-38 °C, which means that the reducing agent may be added at room temperature or at a temperature suitable for fermentation. Therefore, extra process steps for adjusting the temperature may not be required.
  • Thermophilic enzymes may function at temperatures up to 80 °C and in such cases the reducing agent may be added in connection with the hydrolysis reaction, such as to the vessel in which hydrolysis is performed.
  • the least one reducing agent is added at a pH of 3-8, such as 3-6, such as 4-6, such as 5-6.
  • the reducing agent may be added at a pH that is suitable for hydrolysis and/or fermentation. For example, fermentation and SSF is often performed at a pH of about 5.5. Extra process steps for adjusting the pH may therefore not be required.
  • the at least one reducing agent comprises sulphur.
  • the at least one reducing agent may be selected from dithionite and sulphite. These reducing agents have shown to be suitable for decreasing the fermentation inhibition as shown in Examples 1-5 of the present disclosure.
  • Sulphite SO 3 2-
  • Dithionite S 2 O 4 2-
  • the reducing agent may comprise sulphite and/or dithionite in salt form, i.e. complexed with different cations. Examples include Na 2 SO 3 , NaHSO 3 , KHSO 3 , and Na 2 S 2 O 4 .
  • the reducing agent may be dithionite and the dithionite may be added in an amount such that the concentration of dithionite during fermentation is 1-30 mM, such as 5-25 mM, such as 7.5-20 mM.
  • the reducing agent may be sulphite and the sulphite may be added in an amount such that the concentration of sulphite during fermentation is above 10 mM, such as above 15 mM, such as above 20 mM.
  • the amounts of dithionite or sulphite required to achieve a decrease in fermentation inhibitory properties are relatively low and the results from Examples 1-5 of the present disclosure show that such amounts of dithionite or sulphite permit production of high levels of ethanol using either SHF or SSF procedures.
  • it may be more advantageous to add dithionite compared sulphite since dithionite results in a larger decrease in fermentation inhibition compared to sulphite when added to the same concentration, as seen in Examples 1-5 of the present disclosure. Consequently, the same fermentation inhibitory effect may be achieved by addition of a lower concentration of dithionite compared to sulphite. Addition of a lower concentration of dithionite compared to sulphite also means that the total salt concentration during fermentation is lower, which may be beneficial for the fermentation reaction.
  • thiosulphates such as Na 2 S 2 O 3 -5H 2 O and Na 2 S 2 O 3 , alkali- decomposed sugars, ascorbic acid, cysteine, diethanolamine, triethanolamine, dithiothreitol (DTT) and reduced glutathione.
  • the target chemical is ethanol.
  • Ethanol is a target chemical that is derivable from cellulosic biomass and which can be produced by means of fermentation.
  • the target chemical may also be butanol or succinic acids, which are also derivable from cellulosic material.
  • Other examples of target chemicals are other alcohols or acids, alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers, proteins, peptides, amino acids, vitamins, antibiotics and other pharmaceuticals.
  • the fermentation of hydrolysed material may be performed by a fermenting organism, which refers to an organism that is capable of fermenting saccharides into a target chemical.
  • the fermenting organism may be at least one eukaryotic or prokaryotic microorganism, such as bacteria and/or yeast. Examples of bacteria and yeasts which are capable of fermenting saccharides into other chemical compounds are known to the skilled person.
  • Yeasts from Saccharomyces, Pichia and Candida may be used as the fermenting organism.
  • the fermenting organism may for example be wild type, mutant or recombinant Saccharomyces cerevisiae. Using S. cerevisiae for producing a target chemical by means of fermentation is advantageous since S . cerevisiae is well established with regard to industrial fermentation and provides for a high product yield.
  • the material to which the reducing agent is added has a suspended solids content of at least 5 % (w/w), such as at least 10 % (w/w), such as at least 12 % (w/w).
  • the reducing agent may be added to a cellulosic material having relatively high solids content, such as the cellulosic material subjected to SSF or CBP.
  • a cellulosic material having relatively high solids content such as the cellulosic material subjected to SSF or CBP.
  • This enables detoxification or conditioning of pretreated slurry of cellulosic material without any solids separation step.
  • Such in situ detoxification or conditioning provides for a high product yield and cost-efficient recovery of the target chemical, for example through distillation.
  • the material to which the reducing agent is added has a sugar concentration of at least 45 g/l, such as at least 65 g/l, such as at least 85 g/l.
  • the reducing agent may be added to a cellulosic material having a high sugar concentration, such as to the hydrolysed cellulosic material before or during the fermentation in a SHF process.
  • sucrose refers to fermentable saccharides, such as a fermentable monosaccharides and disaccharides.
  • the method according to the first aspect of the invention is further comprising measuring the fermentability of the fermentation of hydrolysed material; and if the measured fermentability is below a reference value, adding at least one reducing agent to the fermentation.
  • the "fermentability" of a fermentation is any parameter that is proportional to the result of the fermentation process.
  • the fermentability may be the sugar consumption rate, the amount of produced target chemical, the produced target chemical yield on consumed sugars and/or the volumetric target chemical productivity.
  • the sugar consumption rate may be measured as the decrease of sugar concentration per hour
  • the amount of target chemical may be measured as g target chemical per liter
  • the produced target chemical yield on consumed sugars may be measured as the number of target chemical molecules produced by each consumed saccharide molecule by monitoring the decrease in saccharide concentration and the increase of target chemical concentration during fermentation
  • the volumetric target chemical productivity may be measured as g target chemical per liter and hour.
  • the fermentability may be measured by measuring the total sugar concentration. If for example the fermenting organism becomes less effective in a SSF process, an increase of total sugar concentration may be measured.
  • the fermentability may also be the inverse value of the total sugar concentration.
  • this offers the possibility of "rescuing" a fermentation process that in some way does not function properly.
  • the glucose consumption rate may be continuously monitored in a fermentation process and if the rate is below a satisfactory reference level, a reducing agent may be added in order to increase the glucose consumption rate.
  • the reference value of the fermentability may for example be selected such that a fermentation process having a fermentability below the reference value, such as below a certain glucose consumption rate, leads to an unsatisfactory amount of target chemical, and a fermentation process with having a fermentability above the reference value, such as above a certain glucose consumption rate, leads to a desired amount of target chemical.
  • a fermentation process having a fermentability below the reference value such as below a certain glucose consumption rate
  • a fermentation process with having a fermentability above the reference value such as above a certain glucose consumption rate
  • the fermentation capacity in a fermentation that has been subjected to the inhibitors for a longer time may not completely recover even though the reducing agent is added. Without being bound by any scientific theory, the inventors believe that this may be due to that part of the yeast dies.
  • extra yeast may be added in addition the reducing agent.
  • the extra yeast may be added before, concurrently or after the reducing agent.
  • the yeast and the reducing agent may be added within two hours, such as within one hour or 30 minutes.
  • the method is further comprising recirculating process water obtained after the production of the target chemical to any step in the production of the target chemical.
  • Recirculation process water refers to reusing process water upstream in the process for producing a target chemical.
  • part or all of the fermentation broth may be recirculated.
  • part or all of the stillage e.g. a filtrate of the stillage
  • the recirculated process water may for example be used as a pretreatment fluid in a pretreatment of cellulosic material, as a hydrolysing liquid in a hydrolysis of cellulosic material or as a fermentation liquid in a fermentation of sugars.
  • dithionite for decreasing the fermentation inhibitory properties of a material being subjected to simultaneous enzymatic hydrolysis and fermentation or a hydrolysate derived from a enzymatic hydrolysis being subjected to fermentation.
  • Dithionite has been found to be advantageous for decreasing the fermentation inhibitory properties of a material in a simultaneous enzymatic hydrolysis and fermentation process, such as in a SSF or CBP.
  • the dithionite may be used prior to or during the simultaneous enzymatic hydrolysis and fermentation process.
  • the material may be a cellulosic material, such as a lignocellulosic material.
  • Example 1 Detoxification of hydrolysate in a separate hydrolysis and fermentation (SHF) process
  • Lignocellulose hydrolysates were produced from spruce wood and sugarcane bagass through thermochemical pretreatment and subsequent enzymatic hydrolysis.
  • sugarcane bagass For SHF experiments with sugarcane bagass, one kg (dry weight, DW) of dried sugarcane bagass was impregnated with 500 g of dilute sulfuric acid (4%) and kept in a plastic bag for 20 h. The impregnated sugarcane bagass was then loaded into a 30-liter reactor. The material was treated with steam at a temperature of 195 °C and a pressure of 14.1 bar during 15 min. The pretreated material, hereafter referred to as the sugarcane bagass slurry, was cooled and stored at 4 °C until further use.
  • sugarcane bagass for SSF experiments was perfomed in the Swedish cellulosic ethanol pilot plant (operated by SEKAB E-Technology, ⁇ rnsköldsvik, Sweden).
  • Sugarcane bagass was treated in a continuous mode in a 30-litre reactor at a temperature of 198-199 °C and with a residence time of 13-14 min.
  • the feed rate was 24 kg (dry weight) per h and the bagass was impregnated with sulfur dioxide (0.5 kg/h).
  • the pH after pretreatment was 2.7.
  • the dry matter content was 19 %.
  • the pretreated material was cooled and stored at 4 °C until further use.
  • the pretreatment of spruce was also performed by SEKAB E-Technology in the Swedish cellulosic ethanol pilot plant. Unbarked wood chips were treated in a continuous mode with sulfur dioxide in a 30-litre reactor at a temperature of 203 °C and with a residence time of 5 min. One kg of sulfur dioxide per 40 kg of wood chips was used. The pH after pretreatment was 2.0-2.3. The dry matter content was 25-27 %. The pretreated material was cooled and stored at 4 °C until further use. The pH of the bagass slurry was adjusted to 5.3 with a 5 M solution of sodium hydroxide. The slurry was then filtered and part of the liquid fraction was discarded to give the slurry a dry-matter content of 10 %.
  • the cellulase preparation which was from Trichoderma reesei ATCC 26921, had a stated activity of 700 endoglucanase units (EGU)/g (Sigma-Aldrich, Steinheim, Germany) and the loading was 319 EGU/g of solids (DW).
  • the cellobiase preparation Novozyme 188, had a stated activity of 250 cellobiase units (CBU)/g (Sigma-Aldrich) and the loading was 23 CBU/g of solids (DW).
  • the enzyme dosages were based on the results of a set of small-scale experiments. After addition of enzymes, the slurries were incubated with shaking (Infors Ecotron, Infors AG, Bottmingen, Switzerland) at 50 °C and 70 rpm for 48 h.
  • sugarcane bagass and spruce hydrolysate were adjusted to pH 2.0 with a 12 M solution of hydrochloric acid to prevent microbial growth during storage.
  • the sugarcane bagass hydrolysate was concentrated by evaporation (Rotavapor Büchi 001, Büchi Labortechnik AG, Flawil, Switzerland) to obtain a similar glucose concentration as in the spruce hydrolysate.
  • the hydrolysates were stored at 4 °C until further use.
  • the pH of the sugarcane bagass and spruce hydrolysates were adjusted to 5.5 with a 5 M solution of sodium hydroxide.
  • the conditioning of each hydrolysate was performed in eight 100-mL glass vessels equipped with magnetic stirrer bars. 26 mL hydrolysate was added to all vessels, and the vessels were placed on a magnetic stirrer plate (IKA-Werke, Staufen, Germany). Sodium dithionite (chemical grade; >87%, Merck, Darmstadt, Germany) was added to hydrolysates in the concentrations 5 and 10 mM. Additions of sodium sulphite to 5 and 10 mM were also performed. The additions were made at room temperature (23 °C) and the samples were kept for 10 min with stirring. The experiments were performed in duplicates.
  • the flow rate was 1.0 mL/min and the column temperature was set to 50°C.
  • a Shodex SP-0810 column (7 ⁇ m, 8x300 mm) was used with the same HPLC system.
  • the elution was performed using Milli-Q water at a flow rate of 1.0 mL/min and the column temperature was set to 80°C.
  • YLClarity software (YoungLin, Anyang, Korea) was used for data analysis.
  • Ethanol measurements were performed by using an enzymatic kit (Ethanol UV-method, Boehringer Mannheim GmbH, Mannheim, Germany). Fermentation experiments were performed to evaluate the effectiveness of the additions and treatments. For comparison, untreated hydrolysates were included in the fermentation experiments as well as reference fermentations of sugar-based medium with an amount of fermentable sugars (i.e. glucose and mannose) corresponding to that in the hydrolysate samples.
  • the fermentations were carried out using baker's yeast (Saccharomyces cerevisiae) (JITAbolaget AB, Rotebro, Sweden).
  • the yeast inoculum was prepared in 750-mL cotton-plugged shake flasks with 300 mL YEPD medium (2% yeast extract, 1 % peptone, 2% D-glucose). The flasks were inoculated and incubated with agitation at 30°C for approximately 12 h. The cells were harvested in the late exponential growth phase by centrifugation (Hermla Z206A, Hermle Labortechnik GmbH, Wehingen, Germany) at 1,500 g for 5 min. The cells were resuspended in an appropriate amount of sterile water to achieve an inoculum consisting of 2.0 g/L (cell dry weight) in all fermentation vessels.
  • YEPD medium 2% yeast extract, 1 % peptone, 2% D-glucose
  • the fermentation was carried out in 14 25-mL glass flasks equipped with magnets for stirring and sealed with rubber plugs pierced with cannulas for letting out carbon dioxide.
  • the hydrolysate samples (23.75 mL), or alternatively the sugar solution for reference fermentations, were added to the fermentation flasks along with 0.5 mL of a nutrient solution (150 g/L yeast extract, 75 g/L (NH 4 ) 2 HPO 4 , 3.75 g/L MgSO 4 7 H 2 O, 238.2 g/L NaH 2 PO 4 . H 2 O), and 0.75 mL of yeast inoculum.
  • a nutrient solution 150 g/L yeast extract, 75 g/L (NH 4 ) 2 HPO 4 , 3.75 g/L MgSO 4 7 H 2 O, 238.2 g/L NaH 2 PO 4 . H 2 O
  • the flasks were incubated at 30°C in a water bath with magnetic stirring (IKA-Werke). Samples for measurement of sugars and ethanol were withdrawn during the fermentation. The glucose levels during the fermentation were estimated by using a glucometer (Glucometer Elite XL, Bayer AG, Leverkusen, Germany).
  • the concentrations of monosaccharides were not affected by the additions of dithionite or sulphite (Tables 1 and 2).
  • the alkali detoxification i.e. the addition of NH 4 OH
  • the glucose concentration of the spruce hydrolysate after alkali detoxification was only about 70 g/L, whereas addition of dithionite or sulphite led to glucose concentrations above 80 g/L.
  • Alkali detoxification also led to smaller amounts of xylose, galactose and mannose in the spruce hydrolysate compared to when dithionite or sulphite were used.
  • the sugarcane bagass hydrolysate was not as inhibitory as the spruce hydrolysate, since there was a steady consumption of glucose, which was depleted in the sample taken after 28 h of fermentation ( Fig. 3 ).
  • the differences between the effects of the various treatments were therefore less pronounced in the sugarcane bagass hydrolysates, but they follow the same pattern as observed in the spruce hydrolysate.
  • volumetric ethanol productivities for samples treated by addition of dithionite or by ammonium hydroxide detoxification rose significantly and were higher than the corresponding values for the reference fermentation (Table 3).
  • Example 1 shows that the addition of reducing agents radically improved the fermentability of inhibitory lignocellulose hydrolysates in the SHF process without the need for a separate detoxification step.
  • Example 2 Detoxification of hydrolysate in a simultaneous saccharification and fermentation (SSF) process
  • yeast inoculum was prepared according to Example 1 above. Inoculums were added to give a start concentration of 2.0 g/L (cell dry weight) in every flask. No source of extra nutrients was added. For comparison, two flasks with spruce slurry to which no reducing agents had been added were included in the experiment. The flasks were incubated at 35°C for 69 h in a water bath with magnetic stirring. The flasks were sealed with Parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA) to prevent evaporation of ethanol. Samples were withdrawn for analysis of ethanol according to Example1 above.
  • Parafilm Parafilm
  • Fig.5 clearly shows that addition of dithionite and sulphite led to higher ethanol production compared to the untreated hydrolysate.
  • the ethanol formation in the samples with 10 mM dithionite leveled off after about 45 h.
  • the samples with 7.5 mM dithionite reached the same high levels of ethanol, but ethanol formation was slightly slower.
  • Ethanol formation in the samples to which sulphite was added leveled off after 20 h and resulted in a lower ethanol production compared to the samples to which dithionite was added ( Fig. 5 ).
  • Example 2 shows that the addition of reducing agents radically improved the fermentability of inhibitory lignocellulose hydrolysates also in the SSF process without the need for a separate detoxification step.
  • Dithionite and sulphite were chosen for the SSF process considering their utilization in large-scale industrial processes.
  • Example 3 Detoxification of spruce slurry in a simultaneous saccharification and fermentation (SSF) process: Ethanol yield as a function of dithionite and sulphite concentrations
  • a spruce slurry was prepared according to Examples 1 and 2 above. Different flasks were filled with 100 g each of the spruce slurry and were subjected to a SSF process according to Example 2 above, but with different amounts of dithionite and sulphite added. The produced ethanol was monitored during the fermentation.
  • Sodium dithionite was added such that the final concentration during fermentation was between 2.5-30 mM. Further, sodium sulphate was added such that the final concentration during fermentation was between 2.5-30 mM.
  • the produced ethanol as a function of time is plotted in Fig. 6 and Fig. 7 . It was seen that treatment with 7.5 mM, 10 mM and 15 mM sodium dithionite resulted in the highest ethanol production, but also 5 mM and 20 mM sodium dithionite resulted in high ethanol concentrations, about 35-40 g/L after 70 hours (see Fig. 6 ).
  • Example 3 shows that a higher ethanol concentration is obtained during fermentation if dithionite is added to a final concentration of 7.5-20 mM compared to if dithionite is added to final concentrations that are outside this range. Further, Example 3 shows that a sulphite addition of above 10 mM is more advantageous, i.e. leading to higher ethanol concentrations during fermentation, compared to if sulphite is added to a final concentration of below 10 mM.
  • Example 4 Detoxification of spruce slurry in a simultaneous saccharification and fermentation (SSF) process: Ethanol yield vs. time of addition of sulphite or dithionite
  • a spruce slurry was prepared as described in Example 1 above. Fermentation experiments with the yeast Saccharomyces cerevisiae were carried out as in the previously described Examples, except that the addition of reducing agent 10 min prior to inoculum was compared with simultaneous addition of reducing agent and inoculum, and addition of reducing agent 45, 105, 240, or 480 min after inoculum. Both sodium dithionite and sodium sulphite were included in the experiments and both were added to 10 mM. The ethanol concentration was determined after 24 hours according to the analysis described in Example 1 above.
  • Example 4 demonstrates that a fermentation in which the ethanol production is inhibited may be "rescued" after the fermentation reaction has been initiated by the addition of a reducing agent.
  • addition of a reducing agent before or simultaneous as the fermentation is initiated results in a higher ethanol yield.
  • specific times used in this lab-scale experiment does not necessarily correspond to an industrial context. Thus additions of reducing agent more than 4 hours after the yeast addition may be efficient in a large scale fermentation.
  • Example 5 Detoxification of sugarcane bagass slurry in a simultaneous saccharification and fermentation (SSF) process: Ethanol yield as a function of dithionite and sulphite concentrations

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
EP09180193A 2009-12-21 2009-12-21 Détoxication avec des agents réducteurs Withdrawn EP2336342A1 (fr)

Priority Applications (12)

Application Number Priority Date Filing Date Title
EP09180193A EP2336342A1 (fr) 2009-12-21 2009-12-21 Détoxication avec des agents réducteurs
US13/517,284 US8795997B2 (en) 2009-12-21 2010-12-17 Detoxification with reducing agents
CA2783200A CA2783200C (fr) 2009-12-21 2010-12-17 Detoxication avec agents reducteurs
AP2012006373A AP3315A (en) 2009-12-21 2010-12-17 Detoxification with reducing agents
CA3035054A CA3035054C (fr) 2009-12-21 2010-12-17 Detoxication avec agents reducteurs
PL10795363T PL2516659T3 (pl) 2009-12-21 2010-12-17 Detoksykacja za pomocą środków redukujących
BR112012015508-3A BR112012015508B1 (pt) 2009-12-21 2010-12-17 método para diminuição da inibição de fermentação em um processo para produzir um produto químico alvo a partir de um material celulósico pré-tratado e uso de ditionita
PCT/EP2010/070127 WO2011080129A2 (fr) 2009-12-21 2010-12-17 Détoxication avec agents réducteurs
EP10795363.0A EP2516659B1 (fr) 2009-12-21 2010-12-17 Détoxication avec des agents réducteurs
CN201080064339.8A CN102770547B (zh) 2009-12-21 2010-12-17 使用还原剂的解毒
EP13194379.7A EP2703493B1 (fr) 2009-12-21 2010-12-17 Détoxication avec des agents réducteurs
ZA2012/04178A ZA201204178B (en) 2009-12-21 2012-06-07 Detoxification with reducing agents

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP09180193A EP2336342A1 (fr) 2009-12-21 2009-12-21 Détoxication avec des agents réducteurs

Publications (1)

Publication Number Publication Date
EP2336342A1 true EP2336342A1 (fr) 2011-06-22

Family

ID=42173882

Family Applications (3)

Application Number Title Priority Date Filing Date
EP09180193A Withdrawn EP2336342A1 (fr) 2009-12-21 2009-12-21 Détoxication avec des agents réducteurs
EP13194379.7A Active EP2703493B1 (fr) 2009-12-21 2010-12-17 Détoxication avec des agents réducteurs
EP10795363.0A Active EP2516659B1 (fr) 2009-12-21 2010-12-17 Détoxication avec des agents réducteurs

Family Applications After (2)

Application Number Title Priority Date Filing Date
EP13194379.7A Active EP2703493B1 (fr) 2009-12-21 2010-12-17 Détoxication avec des agents réducteurs
EP10795363.0A Active EP2516659B1 (fr) 2009-12-21 2010-12-17 Détoxication avec des agents réducteurs

Country Status (9)

Country Link
US (1) US8795997B2 (fr)
EP (3) EP2336342A1 (fr)
CN (1) CN102770547B (fr)
AP (1) AP3315A (fr)
BR (1) BR112012015508B1 (fr)
CA (2) CA3035054C (fr)
PL (1) PL2516659T3 (fr)
WO (1) WO2011080129A2 (fr)
ZA (1) ZA201204178B (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4023762A1 (fr) * 2020-12-29 2022-07-06 Sekab E-Technology AB Procédé et système de réduction de substances inhibitrice d'un matériau à base de biomasse lignocellulosique

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9670510B2 (en) 2011-09-13 2017-06-06 Novozymes A/S Methods of hydrolyzing and fermenting cellulosic material
CN104204213A (zh) * 2012-01-29 2014-12-10 诺维信公司 用于降解或转化纤维素材料的方法
WO2013110242A1 (fr) * 2012-01-29 2013-08-01 Novozymes A/S Procédés de dégradation ou de conversion de matière cellulosique
CN103014098B (zh) * 2012-12-24 2015-01-28 山东大学 一种利用还原剂解除中水对酶解木质纤维素抑制的方法
JP6344388B2 (ja) 2013-07-02 2018-06-20 三菱ケミカル株式会社 糖液の処理方法、水素化処理糖液、有機化合物の製造方法および微生物の培養方法
US9410217B2 (en) 2014-02-18 2016-08-09 Renmatix, Inc. Method of reducing a fermentation and/or enzyme inhibitor in a saccharide-containing composition
WO2015137467A1 (fr) 2014-03-13 2015-09-17 三菱化学株式会社 Procédé de traitement de mélasse, procédé de fabrication de mélasse traitée, mélasse traitée, procédé de fabrication de composé organique, et procédé de culture de microorganisme
US20160145542A1 (en) * 2014-11-25 2016-05-26 Milliken & Company Cleaning Composition
CN110628833A (zh) * 2019-10-24 2019-12-31 中国科学院成都生物研究所 一种以秸秆为原料添加硫代硫酸钠发酵生产丁醇的方法
EP3812466A1 (fr) 2019-10-24 2021-04-28 Sekab E-Technology AB Procédé de formation d'un hydrolysat stable au stockage à partir d'un matériau lignocellulosique

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2094907A4 (fr) 2006-12-18 2012-02-01 Novozymes North America Inc Détoxification de matériaux prétraités contenant de la lignocellulose
BRPI0817541A2 (pt) * 2007-10-12 2014-11-25 Danisco Us Inc Métodos e composições para a produção otimizada de substâncias orgânicas de micro-organismos de fermentação

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ALRIKSSON ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 129-132, 2006, pages 599 - 611
JOÃ Â GBP O R M ALMEIDA ET AL: "Metabolic effects of furaldehydes and impacts on biotechnological processes", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 82, no. 4, 31 January 2009 (2009-01-31), pages 625 - 638, XP019705519, ISSN: 1432-0614 *
LARSSON ET AL: "Comparison of Different Methods for the Detoxification of Lignocellulose Hydrolyzates of Spruce", APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, THE HUMANA PRESS, INC, US LNKD- DOI:10.1385/ABAB:77:1-3:91, vol. 77-79, 1 January 1999 (1999-01-01), pages 91 - 103, XP007904792, ISSN: 0273-2289 *
MILLER ELLIOT N ET AL: "Furfural Inhibits Growth by Limiting Sulfur Assimilation in Ethanologenic Escherichia coli Strain LY180", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 75, no. 19, October 2009 (2009-10-01), pages 6132 - 6141, XP002584225 *
NILVEBRANT ET AL., APPL. BIOCHEM. BIOTECHNOL., vol. 91-93, 2001, pages 35 - 49
OLSSON L ET AL: "Kinetics of ethanol production by recombinant Escherichia coli KO11", BIOTECHNOLOGY AND BIOENGINEERING, vol. 45, no. 4, 1995, pages 356 - 365, XP002584227, ISSN: 0006-3592 *
PALMQVIST EVA ET AL: "Fermentation of lignocellulosic hydrolysates. I: Inhibition and detoxification", BIORESOURCE TECHNOLOGY, vol. 74, no. 1, August 2000 (2000-08-01), pages 17 - 24, XP002584226, ISSN: 0960-8524 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4023762A1 (fr) * 2020-12-29 2022-07-06 Sekab E-Technology AB Procédé et système de réduction de substances inhibitrice d'un matériau à base de biomasse lignocellulosique
WO2022144216A1 (fr) * 2020-12-29 2022-07-07 Sekab E-Technology Ab Procédé et système de réduction de substances inhibitrices d'un matériau à base de biomasse lignocellulosique

Also Published As

Publication number Publication date
EP2703493A1 (fr) 2014-03-05
CA3035054A1 (fr) 2011-07-07
CA3035054C (fr) 2020-08-25
BR112012015508A2 (pt) 2021-02-17
PL2516659T3 (pl) 2014-08-29
CN102770547A (zh) 2012-11-07
BR112012015508B1 (pt) 2021-07-06
AP3315A (en) 2015-06-30
CN102770547B (zh) 2015-04-29
CA2783200C (fr) 2019-07-02
AP2012006373A0 (en) 2012-08-31
WO2011080129A2 (fr) 2011-07-07
ZA201204178B (en) 2013-08-28
WO2011080129A3 (fr) 2011-09-01
EP2703493B1 (fr) 2016-04-13
EP2516659A2 (fr) 2012-10-31
EP2516659B1 (fr) 2014-03-05
CA2783200A1 (fr) 2011-07-07
US20130011894A1 (en) 2013-01-10
US8795997B2 (en) 2014-08-05

Similar Documents

Publication Publication Date Title
EP2524048B1 (fr) Détoxication in situ d'inhibiteurs de fermentation avec des agents réducteurs
EP2516659B1 (fr) Détoxication avec des agents réducteurs
EP2625279B1 (fr) Hydrolyse enzymatique de matériau lignocellulosique en présence d'oxyanions du soufre et/ou de composés de sulfhydryle
Alriksson et al. Improving the fermentability of enzymatic hydrolysates of lignocellulose through chemical in-situ detoxification with reducing agents
US8110383B2 (en) Fermentation process starting from cellulosic biomass and involving the recirculation of detoxified stillage into the process
Survase et al. Continuous acetone–butanol–ethanol fermentation using SO2–ethanol–water spent liquor from spruce
EP2726623B1 (fr) Méthodes pour l'amélioration de l'hydrolyse enzymatique des matières lignocellullosiques
van Groenestijn et al. Production of ethanol from wheat straw by pretreatment and fermentation at high dry matter concentrations
Rana et al. Enzyme Production from Trichoderma reesei and Aspergillus Strain

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

AX Request for extension of the european patent

Extension state: AL BA RS

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20111223